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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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Elevated ozone reduces photosynthetic carbon gain by accelerating leaf senescence of
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inbred and hybrid maize in a genotype-specific manner
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Running title: Maize photosynthetic response to ozone
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Craig R. Yendrek1#, Gorka Erice1*, Christopher M. Montes1,2, Tiago Tomaz1‡, Crystal A.
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Sorgini1,3, Patrick J. Brown1,3, Lauren M. McIntyre4,5, Andrew D.B. Leakey1,2,3, Elizabeth A.
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Ainsworth1,2,3,6
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10
1
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Urbana, IL, 61801
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2
Department of Plant Biology, University of Illinois at Urbana Champaign, Urbana, IL, 61801
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Department of Crop Sciences, University of Illinois at Urbana Champaign, Urbana, IL 61801
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Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL,
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32610
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Genetics Institute, University of Florida, Gainesville, FL, 32610
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USDA ARS, Global Change and Photosynthesis Research Unit, Urbana, IL, 61801
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#
current address: The Scotts Company, Marysville, OH 43041
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*current address: Estación Experimental del Zaidín (EEZ-CSIC), Spain
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‡current address: Sun Pharmaceuticals Industries, Latrobe, Tasmania
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana Champaign,
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Corresponding author: Elizabeth Ainsworth, 1201 W. Gregory Drive, 147 ERML, Urbana, IL
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61801 USA, [email protected])
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ABSTRACT
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Exposure to elevated tropospheric ozone concentration ([O3]) accelerates leaf senescence in
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many C3 crops. However, the effects of elevated [O3] on C4 crops including maize (Zea mays L.)
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are poorly understood in terms of physiological mechanism and genetic variation in sensitivity.
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Using Free Air gas Concentration Enrichment (FACE), we investigated the photosynthetic
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response of 18 diverse maize inbred and hybrid lines to season-long exposure to elevated [O3]
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(~100 nL L-1) in the field. Gas exchange was measured on the leaf subtending the ear throughout
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the grain filling period. On average over the lifetime of the leaf, elevated [O3] led to reductions
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in photosynthetic CO2 assimilation of both inbred (-22%) and hybrid (-33%) genotypes. There
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was significant variation among both inbred and hybrid lines in the sensitivity of photosynthesis
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to elevated [O3], with some lines showing no change in photosynthesis at elevated [O3]. Based
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on analysis of inbred line B73, the reduced CO2 assimilation at elevated [O3] was associated with
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accelerated senescence decreasing photosynthetic capacity, and not altered stomatal limitation.
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These findings across diverse maize genotypes could advance the development of more ozone
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tolerant maize, and provide experimental data for parameterization and validation of studies
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modeling how O3 impacts crop performance.
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Keyword index
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Zea mays, Photosynthesis, Senescence, Stomatal conductance, Tropospheric ozone
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INTRODUCTION
Tropospheric ozone (O3) is an airborne pollutant that enters leaves through their stomata
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and generates reactive oxygen species (ROS) upon contact with intercellular leaf surfaces (Heath
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1988). Subsequently, these ROS can cause oxidative damage to membranes and other cellular
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components. At very high concentrations, O3 can elicit a hypersensitive response, but even at
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lower concentrations O3 can impair cellular function (Krupa & Manning 1988). When exposed to
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elevated O3 concentrations ([O3]) throughout the growing season, many plants have reduced
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photosynthetic C assimilation and biomass production as well as increased antioxidant capacity
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and mitochondrial respiration rates (Fiscus et al. 2005; Ainsworth et al. 2012). As a cumulative
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result of these physiological responses, crops exhibit significant reductions in economic yield
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with increasing [O3] (Mills et al. 2007; Ainsworth 2017).
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The effects of exposure to chronic, elevated [O3] on photosynthesis and stomatal
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conductance can vary with leaf age and canopy position (Tjoelker et al. 1995; Morgan et al.
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2004; Betzelberger et al. 2010; Feng et al. 2011). Studies that repeatedly measured a cohort of
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soybean and wheat leaves found that photosynthesis and stomatal conductance were not
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significantly affected by elevated [O3] in recently mature leaves, but continued exposure over
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time accelerated the decline in photosynthetic capacity as leaves aged (Morgan et al. 2004; Feng
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et al. 2011; Emberson et al., 2017). Stomatal conductance can also become uncoupled from
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photosynthesis as leaves age under O3 stress, often with greater measured reductions in
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photosynthesis than stomatal conductance (Lombardozzi et al. 2012), resulting in decreased
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water use efficiency at elevated [O3] (Tjoelker et al. 1995; VanLoocke et al. 2012). Additionally,
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stomata of plants exposed to elevated [O3] can show delayed opening or closing responses to
3
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environmental or hormonal signals (Keller & Häsler 1984; Wilkinson & Davies 2009; Paoletti &
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Grulke 2010; Wagg et al. 2013).
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Much of our understanding of the effects of elevated [O3] on photosynthesis and stomatal
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function come from study of C3 species (Reich 1987; Paoletti & Grulke 2005; Felzer et al. 2007;
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Wittig et al. 2007). A few experiments have shown that elevated [O3] can negatively impact
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photosynthetic properties and biomass production of the model C4 species, maize (Zea mays L.)
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(Pino et al. 1995; Leitao et al. 2007a; Leitao et al. 2007b; Bagard et al. 2015; Yendrek et al.
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2017). However, current understanding of the mechanisms underlying photosynthetic responses
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to O3 in maize is limited to a few genotypes, and often to juvenile growth stages. The high ratio
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of photosynthetic CO2 assimilation relative to stomatal conductance and isolation of Rubisco
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carboxylation in the bundle sheath cells make it possible that C4 photosynthesis will respond
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distinctly to C3 photosynthesis at elevated [O3]. Analysis of historical U.S. maize yields suggests
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that reported physiological responses to O3 pollution drive annual yield reductions of
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approximately 10% from 1980-2011, amounting to $7.2 billion in lost profit per year on average
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(McGrath et al. 2015). Considering that O3-mediated yield reductions were more prominent in
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hot and dry years (McGrath et al. 2015) and future climate models predict an increase in growing
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season temperature with altered frequency and magnitude of precipitation events (Christensen et
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al. 2007; Walsh et al. 2014), efforts to address the knowledge gap about mechanisms of O3-
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sensitivity in maize are likely to become increasingly important.
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In addition to being the most widely produced food crop, maize is a model C4 species
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with considerable resources for analysis of genotype to phenotype associations. This includes the
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maize nested association mapping (NAM) population, which was developed to encompass much
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of the existing diversity in a relatively small number of inbred lines (Yu et al. 2008). Substantial
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phenotypic diversity for many agronomic traits exists within the parental lines of the NAM
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(Flint-Garcia et al. 2005). In order to exploit these resources to locate genetic factors associated
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with complex traits such as O3 tolerance, there is a need to first identify lines that respond
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differently to elevated [O3] for key physiological traits. For understanding and modeling the
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impacts of O3 on crops, photosynthesis and stomatal conductance are two key traits (Reich 1987;
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Emberson et al. 2000; Sitch et al. 2007). In this study, we investigated the effects of season-long
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growth at elevated [O3] on maize photosynthetic gas exchange. Maize was exposed to elevated
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[O3] in the field using Free Air gas Concentration Enrichment (FACE). In 2013 and 2014, we
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studied the inbred cultivar B73. We hypothesized that growth at elevated [O3] would reduce
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photosynthetic capacity and daily C gain as leaves progressed more rapidly through senescence.
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In 2015, we investigated the response of photosynthesis and stomatal conductance to elevated
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[O3] in 10 diverse inbred and 8 diverse hybrid lines in order to test for genetic variation in O3
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response.
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MATERIALS AND METHODS
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Field site and experimental conditions
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Maize (Zea mays) inbred and hybrid lines (Table 1) were studied at the FACE research
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facility in Champaign, IL (www.igb.illinois.edu/soyface/) in 2013, 2014 and 2015. This facility
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is located on a 32 ha farm. Maize is grown on half of the land each year, and rotated annually
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with soybean. Each year, the portion of the field growing maize was fertilized with N (180 lbs
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per acre) and treated with pre-emergent and post-emergent herbicides at the recommended rates.
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Additional weeding was done inside the FACE rings as needed. Maize inbred and hybrid lines
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were planted with a precision planter in rows spaced 0.76 m apart and 3.35 m in length, at a
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density of 8 plants m-1 in replicated blocks (n=4). Each block had one ambient plot and one
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elevated [O3] plot, which were octagonal in shape and of 20-m diameter. Weather conditions
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were monitored at the FACE facility, and Supplemental Figure 1 shows the seasonal time
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courses of: (1) daily maximum, (2) daily minimum temperatures, and (3) daily total precipitation
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for the 2013, 2014 and 2015 growing seasons.
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Air enriched with O3 was delivered to the experimental rings with FACE technology, as
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described in Yendrek et al. (2017). The target fumigation set-point in the elevated [O3] rings was
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100 nL L-1, which was imposed for ~8 h per d throughout the growing season except when
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leaves were wet or wind speeds were too low to ensure accurate fumigation (i.e., <0.5 m s-1).
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Over the three years, when the fumigation was on, the averaged 1-min [O3] within the treatment
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rings was within 10% of the 100 nL L-1 set point for 53.7% of the time and within 20% of the set
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point for 78% of the time. Season-long 8 hr average [O3] in ambient rings was 40.8 nL L-1 in
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2013, 40.1 nL L-1 in 2014, and 40.0 nL L-1 in 2015, and season-long elevated [O3] was 71.0 nL
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L-1 in 2013, 70.8 nL L-1 in 2014 and 64.0 nL L-1 in 2015.
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Leaf-level gas exchange measurements
In situ photosynthetic gas exchange of the leaf subtending the ear was measured using
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portable photosynthesis systems (LI-6400, LICOR Biosciences, Lincoln, NE) in a modified
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version of a previously published protocol (Leakey et al. 2006). The net rate of photosynthetic
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CO2 assimilation (A), stomatal conductance (gs), the ratio of intercellular [CO2] (ci) to
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atmospheric [CO2] (ca), and instantaneous water use efficiency (iWUE = A/gs) were measured.
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Reproductive development (time to silking and anthesis) was monitored in all experimental
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rows, and all dates of gas exchange measurements are reported relative to the date of anthesis.
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In 2013 and 2014, the diurnal time course of leaf photosynthetic gas exchange was
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assessed with measurements taken across all replicate plots every few hours throughout the day
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at three or four development stages over the growing season of the B73 inbred genotype. In
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2015, in situ gas exchange measurements on 10 inbred and 8 hybrid lines were collected at
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midday approximately every 7 d starting when the plants reached anthesis and ending when the
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ear leaf of >50% plants in an individual row had fully senesced. After senescence of a genotype
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in a given treatment, we recorded zero for A and gs for all subsequent weekly measurements until
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both ambient and elevated plots were fully senesced. In all years, two or three plants were
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measured as sub-samples within each replicate plot at either ambient [O3] or elevated [O3].
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Prior to a set of measurements being performed over a 1-2 h period of a day in all
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replicate plots, temperature and relative humidity (RH) in the leaf chamber cuvette were set to
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match prevailing ambient conditions as measured by an on-site weather station. A linear
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quantum sensor (AccuPAR LP-80; Decagon Devices, Pullman, WA) was used to measure the
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average light intensity at the position in the canopy for the leaf subtending the ear, which was
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used to set the PPFD incident on the leaf in the gas exchange cuvette. In 2015, a constant PPFD
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was used for all weekly midday measurements based on the measured PPFD at anthesis. For
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inbred cultivars, PPFD was set at 1,800 µmol m-2 s-1 and for hybrid cultivars PPFD was set at
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450 µmol m-2 s-1. Keeping light constant over the entire measurement period enabled comparison
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of changes in the response of A and gs to leaf aging in ambient and elevated [O3], and eliminated
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variation in those parameters due to PPFD. On all dates, four instruments were used
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simultaneously. All environmental conditions were held constant across instruments.
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The response of A to ci was measured on the leaf subtending the ear in 2013 and 2014 to
examine the effects of elevated [O3] on the maximum apparent rate of phosphoenolpyruvate
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carboxylase activity (Vpmax) and on CO2-saturated photosynthetic rate (Vmax). Two to three leaves
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per ring were measured. Leaves were excised pre-dawn, immediately re-cut under water, and
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measured in a field laboratory. This approach minimized short-term decreases in water potential,
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chloroplast inorganic phosphate concentration, and maximum PSII efficiency that can occur in
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the field, and also enabled more leaves to be measured on multiple portable photosynthesis
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systems over a short period of time (Leakey et al. 2006). Measurements were initiated at ambient
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ci, then the reference [CO2] in the leaf cuvette was stepped down to 25µmol mol-1, before it was
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increased stepwise to 1200 µmol mol-1 while keeping PPFD constant at 2,000 µmol m-2 s-1. The
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initial slope of the A/ci curve (ci < 60 µmol mol-1) was used to calculate Vpmax according to von
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Caemmerer (2000). A four parameter nonrectangular hyperbolic function was used to estimate
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Vmax as the horizontal asymptote of the A/ci curve. Stomatal limitation to A (l) was estimated
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from A/ci curves using the approach described by Farquhar and Sharkey (1982), and modified by
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Markelz et al. (2011). Mean values of Vpmax and Vmax from A/ci curves were used in combination
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with in situ values of ci measured at midday to estimate the range of l and mean l for recently
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mature leaves (DOY 219 in 2013, DOY 213 in 2014) and senescing leaves (DOY 238 in 2013,
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DOY 245 in 2014) in ambient and elevated [O3].
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Statistical analysis
Diurnal gas exchange parameters (A, gs) measured in 2013 and 2014 were analyzed with
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a repeated mixed model ANOVA with the Kenwood-Rogers option and compound symmetry
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covariance structure (SAS, version 9.4, Cary, NC). All analysis was done on the ring mean. Days
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were analyzed independently, time of day was a repeated measure and block was a random
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effect. A mixed model ANOVA with the Kenwood-Rogers specification was used to analyze
8
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Vpmax and Vmax in 2013 and 2014 (SAS, Version 9.4, Cary, NC). O3 treatment and DOY were
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fixed effects in the model, block was a random effect, and years were analyzed independently.
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Statistical differences in least squared mean estimates were determined by linear contrasts with a
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threshold p<0.05.
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A and gs of the flag leaf measured in 2015 were fit with a quadratic equation: y = yo + x
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+ x2, where y was A or gs, and x was days after anthesis (Proc Nlin, SAS). Other models
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(exponential decay, 3-parameter Weibull function) were tested, but did not fit all of the data for
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each genotype. Therefore, a simple quadratic equation was used. In order to test if there were
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differences in the decline in A or gs over time in ambient and elevated [O3], a single quadratic
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model was first fit to the A and gs data for each genotype, then models were fit to each genotype
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and treatment combination. An F statistic was used to test if the model with genotype and
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treatment produced a significantly better fit to the data, i.e., if there were significant differences
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in the response of A or gs over time in ambient and elevated [O3]. Additionally the quadratic
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model was fit to each genotype within each ring in order to obtain parameter estimates for yo, 
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and Parameter estimates as well as initial values of A and gs measured shortly after anthesis
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were analyzed with a mixed model ANOVA with the Kenwood-Rogers specification. O3
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treatment and genotype were fixed effects in the model and block was a random effect. Statistical
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differences in least squared mean estimates between ambient and elevated [O3] for each inbred or
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hybrid line were determined by linear contrasts with a threshold p<0.05.
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RESULTS
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Elevated [O3] accelerates loss of photosynthetic capacity in inbred line B73
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In 2013 and 2014, daily time courses of leaf photosynthetic gas exchange were measured
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in situ as the leaf subtending the ear aged in ambient and elevated [O3] (Figs. 1, 2). When the leaf
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was recently fully expanded, shortly before or after anthesis, there was no significant effect of
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elevated [O3] on A (Figs. 1d, 2e, 2f). However, as the leaf aged, growth at elevated [O3]
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accelerated the decline in diurnal C gain, and later in the season A was significantly reduced by
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elevated [O3] (Figs. 1e, 1f, 2g, 2h). Significant differences in A between ambient and elevated
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[O3] were apparent earlier in the growing season than differences in gs (Figs. 1e versus 1h, 2g
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versus 2k). But, there were no consistent differences in the ratio of intercellular [CO2] to
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atmospheric [CO2] (ci/ca) or instantaneous water use efficiency (iWUE = A/gs) in ambient and
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elevated [O3] in either year (Fig. 1j-o, Fig. 2m-t). On only one sampling date out of seven, which
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was characterized by low PPFD, elevated [O3] led to significantly greater ci/ca and significantly
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lower iWUE (Fig. 1k, n).
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Declines in in situ A over the leaf aging process were associated with decreased
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photosynthetic capacity, measured as Vpmax (Fig. 3a, b; Table 2) and Vmax (Fig. 3c, d; Table 2).
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Declines in Vpmax from when the leaf was recently fully expanded to until ~30 days later were
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greater in elevated [O3] (-64% in 2013 and -64% in 2014) than in ambient [O3] (-51% in 2013
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and -33% in 2014; Fig. 3a, b). Likewise, declines in Vmax from when the leaf was recently fully
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expanded to until ~30days later were greater in elevated [O3] (-40% in 2013 and -52% in 2014)
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than in ambient [O3] (-25% in 2013 and -33% in 2014; Fig. 3c, d). Although an O3 treatment
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effect was not significant in the mixed ANOVA model (Table 2), pair-wise comparisons of the
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means showed that Vpmax was significantly lower in elevated [O3] on the final sampling date in
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2014 (Fig. 3b) and Vmax was significantly lower in elevated [O3] on the final sampling date in
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both 2013 and 2014 (Fig. 3c, d).
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A/ci curves were also used to assess stomatal vs. biochemical limitations to A in aging
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leaves grown at ambient and elevated [O3]. Average A/ci curves in ambient and elevated [O3]
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were plotted for the leaf subtending the ear shortly after anthesis, and then ~3-4 weeks later (Fig.
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4). Shortly after anthesis, l was minimal in both ambient and elevated [O3] in 2013 (2-3%), and
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mean ci was well above the inflection point of the A/ci curve (Fig. 4a). In 2014, there was a wider
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range of observed ci shortly after anthesis, but mean l was still low (5-6%) in both ambient and
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elevated [O3], and the mean ci was above the inflection point (Fig 4b). As leaves aged, l
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increased in both ambient and elevated [O3] to 20-30% (Fig. 4c, d). Both increased l and
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decreased photosynthetic capacity result in lower A in aging leaves, but elevated [O3] did not
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appear to change l.
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Photosynthetic response to elevated [O3] in diverse inbred and hybrid lines
In 2015, A of the leaf subtending the ear was measured approximately weekly in 10
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diverse inbred lines and 8 hybrid lines during the grain filling period. Initial values of A
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measured shortly after anthesis differed among lines, but not between ambient and elevated [O3],
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and the interaction between inbred line and [O3] was only marginally significant (Table 3).
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Similarly, initial measurements of gs varied among inbred lines, but not between ambient and
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elevated [O3] (Table 3). For 8 of the 10 inbred lines, there was significant acceleration in the
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decline in A as the leaf aged under elevated [O3], similar to what was observed in previous years
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in B73 (Fig. 5). However, for two inbred lines (M37W and CML333), there was no significant
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difference in the quadratic fit to the photosynthetic data between ambient and elevated [O3] (Fig.
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5). There were highly significant effects of [O3], inbred line and their interaction on the
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parameters describing the quadratic equation modeling the decline in A over time in the inbred
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lines (Table 3). This reflected variation in the estimates of A at anthesis (yo), the time to the start
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of a decline in A () and the rate of decline in A (). The significant effect of [O3] on yo is not
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consistent with the lack of an effect on initial rates of A, but some of the data were best fit by
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convex functions and some by concave functions which affected accurate prediction of yo in the
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inbred lines (Fig. 5). Average was 0.30 in ambient [O3] and -0.69 in elevated [O3], indicating
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that a decline in A started earlier in elevated [O3] (Fig. 5, Supplemental Table 1). Average  was
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-0.016 in ambient [O3] and -0.00025 in elevated [O3] indicating that the rate of decline in A was
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greater in ambient [O3] than elevated [O3] on average across inbred lines (Supplemental Table
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1).
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Measurements of gs in the aging flag leaf in inbred lines differed from the response of A
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over time. For 7 of the 10 genotypes, there was no evidence for accelerated decline in gs over
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time (Fig. 6). Of the remaining 3 genotypes, MS71 and C123 displayed more rapid decline in gs
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over time, while NC338 had initially higher gs in elevated [O3]. The influence of this small set of
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genotypes meant that there were significant effects of [O3], inbred line and their interaction on
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the shape of the quadratic fit to the decline in gs over time, especially on parameters  and 
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(Table 3; Supplemental Table 1). Apparent differences in the response of A and gs to elevated
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[O3] over the grain filling period did not impact the iWUE, which was not different in ambient
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and elevated [O3] (Supplemental Fig. 2).
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All 8 of the hybrid lines showed acceleration in the decline of A in the leaf subtending the
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ear over the grain filling period (Fig. 7). Unlike the inbreds, the hybrids had similar initial values
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of A and gs shortly after anthesis (Table 3). These measurements were taken at a lower light level
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than the inbred measurements, which was representative of the light measured in the canopy at
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the leaf subtending the ear in hybrids vs. inbreds, but may explain why there was less variation
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among hybrid lines. There was a significant effect of [O3] and a significant [O3] x hybrid line
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interaction on the parameters describing the decline in A over time (Table 3; Supplemental Table
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1). Consistent with the inbred lines, was positive in ambient [O3] (1.06) and negative in
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elevated [O3] (-0.033), indicating a more immediate decline in A in elevated [O3], and  was
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more negative in ambient [O3] (-0.02) than elevated [O3] (-0.006) indicating more rapid decline
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in A in ambient [O3] (Supplemental Table 1). Only 3 of the 8 hybrid lines showed significant
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changes in gs at elevated [O3] over the grain filling period (Fig. 8), and consistent with the inbred
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lines, the apparent differences in the response of A and gs to elevated [O3] over time did not
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impact iWUE (Supplemental Fig. 3). Notably, the hybrids of MS71 and C123 crossed with B73
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were among this sensitive group, matching the greater than average sensitivity of these
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genotypes as inbreds.
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DISCUSSION
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Tropospheric [O3] continues to increase in many of the world’s crop growing regions as a
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result of inconsistent regulation of precursor pollutants around the globe and short- and long-
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distance transport of pollutants (Lin et al. 2017). Early studies of maize reported that it was
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significantly more tolerant to increasing [O3] than C3 crops like soybean or peanut (Heck et al.
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1983). However, more recent experimental studies using open top chambers reported that maize
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leaf physiology is sensitive to chronic O3 exposure in the cultivar Chambord (Leitao et al. 2007a;
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Leitao et al. 2007b). Furthermore, multiple regression analysis of county-level yield data
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suggests that there is significant yield loss to O3 in the Midwest U.S. growing region (McGrath et
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al. 2015). Here, we examined how season-long fumigation with elevated [O3] using FACE
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technology in the primary area of maize production affected gas exchange of the leaf subtending
13
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294
the ear. We concentrated on this leaf and time period because previous research has established
295
that most of the photosynthate used for grain filling in maize is provided by mid-canopy leaves
296
after anthesis (Borrás et al. 2004), and those leaves are the last on the plant to senesce
297
(Valentinuz & Tollenaar 2004). Diurnal measurements of B73 showed that ear leaf C
298
assimilation was not affected by elevated [O3] around the time of anthesis when the leaf was
299
recently mature; however, loss of photosynthetic capacity of that leaf was accelerated by
300
elevated [O3] (Figs. 1 & 2).
301
The seasonal pattern of elevated [O3] effects on photosynthetic carbon gain over the diurnal
302
period was reflected in measurements of A at midday (Figs. 1 & 2). Therefore, screening of
303
midday A was used to explore genotypic variation in sensitivity to elevated [O3] among diverse
304
hybrid and inbred lines of maize. On average, the sum of midday CO2 assimilation by the leaf
305
subtending the ear over the period from anthesis to complete leaf senescence was reduced by
306
22% in inbred lines and 33% in hybrid lines (Table 4). However, sensitivity to elevated [O3] in
307
inbred lines ranged from no loss in photosynthetic CO2 assimilation (CML333, M37W) to 59%
308
lower CO2 assimilation (C123; Fig. 5, Table 4). Likewise, sensitivity to elevated [O3] in hybrids
309
ranged from 13% lower photosynthetic CO2 assimilation (B73 x M37W) to 44% lower CO2
310
assimilation (B73 x MS71; Fig. 7, Table 4). All ozone sensitive lines showed the same
311
developmentally dependent pattern of response as B73, with similar A in young leaves at
312
ambient and elevated [O3], but more rapid decline in A over time (Fig. 5, 7). Thus, acceleration
313
of senescence rather than decreased initial investment in photosynthetic capacity is a key
314
response of maize to elevated [O3] at the leaf level. But, there is genetic variation in response
315
that might be exploited to breed maize with improved tolerance to ozone pollution, and possibly
316
even broader spectrum oxidative stress tolerance.
14
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
317
Accelerated loss of photosynthetic capacity in elevated [O3] has been documented in FACE
318
experiments with soybean (Morgan et al. 2004; Betzelberger et al. 2010), wheat (Feng et al.
319
2011; 2016), rice (Pang et al. 2009) and now maize. In rice and wheat, accelerated senescence of
320
the flag leaf at elevated [O3] shortened the grain filling duration and reduced seed yields (Gelang
321
et al. 2000; Pang et al. 2009; Feng et al. 2011). Additionally, genetic variation in the response of
322
seed yield to elevated [O3] was attributed in part to variation in leaf senescence (Pang et al. 2011;
323
Feng et al. 2011) as well as to variation in detoxification and antioxidant capacity (Feng et al.
324
2010; 2016). Acceleration of senescence in the ear leaf of maize is associated with maize yield
325
loss under a number of environmental stresses (Wolfe et al. 1988; Bänzinger et al. 1999), and
326
greater yield potential of newer maize varieties has been associated with maintenance of
327
photosynthetic capacity of the ear leaf for a longer period of time following anthesis (Ding et al.
328
2005). Thus, the acceleration of senescence reported here under elevated [O3] for both inbred and
329
hybrid maize has the potential to contribute to grain yield losses.
330
We found that growth at elevated [O3] both accelerated the speed at which flag leaves lost
331
photosynthetic capacity (represented by the  parameter in our quadratic model) and decreased
332
the length of time that leaves maintained optimum photosynthetic capacity ( term in quadratic
333
model). It would be interesting to test the response of maize lines with delayed leaf senescence
334
or stay-green phenotypes for O3 response. There are a variety of genetic and physiological
335
mechanisms that enable a stay-green phenotype (Thomas & Howarth 2000), and there may be
336
mechanisms that are more promising under O3 stress than others. For example, stay-green lines
337
with altered chlorophyll catabolism may be less O3-tolerant as the build-up of phytotoxic
338
degradation products might compete with O3-induced reactive oxygen species for detoxification
339
by the anti-oxidant system. However, stay-green lines with perennial tendencies or lines where
15
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
340
senescence is initiated later, but progresses at the normal rate and with typical catabolism may
341
have the potential to retain higher rates of photosynthesis later in the growing season, and
342
therefore yield better in higher [O3].
343
Studies in a number of C3 species have indicated that biochemical limitation, and not
344
stomatal limitation, are responsible for the sustained effects of elevated [O3] on A (Farage et al.
345
1991; Morgan et al. 2004; Paoletti & Grulke 2005; Feng et al. 2011). In C3 species, accelerated
346
decline in A in aging leaves was associated with decreased maximum carboxylation capacity and
347
electron transport capacity (Zheng et al. 2002; Morgan et al. 2004; Feng et al. 2011). We found a
348
similar response in the maize inbred line B73 with the decline in A at elevated [O3] associated
349
with decreased photosynthetic capacity (Vpmax and Vmax), and not with any change in limitation
350
imposed by stomata. Stomatal limitation increased from 2-5% in the ear leaf when it was
351
recently mature to 20-30% when it was 4-5 weeks older, which is consistent with the loss of
352
stomatal control in aging leaves previously described for other species (Reich 1984). This
353
increase in stomatal limitation occurred in both ambient and elevated [O3], and so there does not
354
appear to be evidence for an uncoupling of A and gs in elevated [O3] that has been described in
355
other studies (Paoletti & Grulke 2005). Across the diverse inbred and hybrid lines grown at
356
elevated [O3], both A and gs declined with ear leaf age (Figs. 5-8), although the reduction in A
357
was often greater than gs. Still iWUE remained relatively constant (Supplemental Fig. 2, 3),
358
providing further evidence from diverse maize lines that growth at elevated [O3] does not result
359
in uncoupling of A and gs.
360
In addition to testing for cultivar variation in response to elevated [O3], we were also able to
361
identify significant variation in A and gs in recently mature flag leaves of inbred maize lines
362
(Table 3). The hybrid lines, which all contained B73 as the female parent, did not show variation
16
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
363
in A and gs (Table 3). Previous studies have also reported that inbred lines show greater variation
364
in photosynthetic traits compared to hybrid lines (Albergoni et al. 1983; Ahmadzadeh et al.
365
2004), but that hybrid lines maintain higher rates of A over a leaf lifespan compared to inbred
366
lines (Ahmadzadeh et al. 2004). We found similar results in our studies of diverse maize lines,
367
however, it should be duly noted that due to the recurrence of B73 as the female parent, there
368
was more limited genetic diversity in the hybrids used in this study. Additionally, the light
369
environment at the ear leaf at anthesis was different in inbred and hybrid canopies. A and gs were
370
monitored at higher PPFD in the inbred lines (1,800 µmol m-2 s-1) compared to the hybrid lines
371
(450 µmol m-2 s-1), which may have also contributed to the greater variation observed amongst
372
inbred lines.
373
Our study showed that accelerated loss of photosynthetic capacity was the basis for
374
photosynthetic sensitivity to elevated [O3] in both inbred and hybrid maize lines. Drought and
375
high temperature stress also negatively impact photosynthesis in maize (Bänzinger et al. 1999;
376
Neiff et al. 2016), and those stresses often co-occur with high O3 events, and will likely increase
377
in the future (Cook et al. 2014). Further experimental studies are needed to understand the
378
interactions of rising [O3] with intensifying drought and heat stress, but our work suggests that a
379
key trait for robust improvement of maize response to elevated [O3] is maintenance of
380
photosynthetic capacity during the grain filling period. By testing a diverse panel of maize
381
genotypes under field conditions in the world’s primary area of production, this study provides a
382
foundation on which to investigate the genetic variation in maize oxidative stress tolerance, and
383
possibly develop more stress tolerant germplasm. Under elevated [O3], both inbred and hybrid
384
maize lines were shown to have reduced carbon gain of the leaf subtending the ear, which
385
heavily influences yield. In contrast to many previous studies of crop responses to atmospheric
17
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
386
change using FACE technology (e.g. Markelz et al. 2011; Gillespie et al. 2012), this study
387
describes the average and variance in treatment effects across diverse genotypes, and so should
388
provide experimental data that is a stronger basis for parameterization or validation of studies
389
attempting to model regional crop performance.
390
391
392
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation (grant no. PGR–1238030)
393
and the USDA ARS. We thank Don Ort for directing the SoyFACE facility, Alvaro Sanz for
394
assistance with gas exchange measurements and Kannan Puthuval, Brad Dalsing and Chad Lantz
395
for management of the FACE rings.
396
397
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
TABLES
Table 1. Planting date (day of year), inbred and hybrid lines examined in each field season, and
the date that anthesis was recorded for each line.
Year
2013
2014
2015
Planting
(DOY)
136, 137
139
139
Line
B73
B73
B73
C123
CML333
Hp301
IL14H
M37W
Mo17
MS71
NC338
OH43
B73xC123
B73xCML333
B73xHP301
B73xIL14H
B73xM37W
B73xMo17
B73xMS71
B73xOH43
Anthesis
(DOY)
208
210
205
203
214
205
201
211
204
202
214
201
202
207
202
201
206
202
201
201
26
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Table 2. Analysis of variance (F, p) of the maximum apparent rate of phosphoenolpyruvate
carboxylase activity (Vpmax) and CO2-saturated photosynthetic rate (Vmax) measured in inbred line
B73 grown at ambient and elevated [O3] in 2013 and 2014. Years were analyzed independently.
2013
Vpmax
Vmax
2014
F
p
F
p
O3
0.39
0.5754
2.46
0.1296
DOY
129
<0.0001
8.57
0.0005
O3 x DOY
2.22
0.1867
0.92
0.4457
O3
2.97
0.1358
3.02
0.1327
DOY
67.9
0.0002
18.3
<0.0001
O3 x DOY
4.01
0.0921
0.94
0.4411
27
Table 3. Analysis of variance (F, p) of photosynthesis (A), stomatal conductance (gs) and the quadratic parameters describing the
decline in A and gs over time. Measurements were made in in 10 inbred and 8 hybrid lines grown at ambient and elevated [O3] in
2015, and inbreds and hybrids were analyzed in separate models. A and gs represent the first measurement taken shortly after anthesis
in Figs. 5-8.
Inbred Lines
A (mol m-2 s-1)
A
gs (mol m-2s-1)
yo

gs

yo


[O3]
1.33, 0.254
0.26, 0.609
13.1, 0.0006
28.8, <0.0001
28.0, <0.0001
2.95, 0.091
6.92, 0.011
7.65, 0.007
Line
2.93, 0.006
2.89, 0.007
12.9, <0.0001
13.0, <0.0001
9.19, <0.0001
8.19, <0.0001
7.67, <0.0001
5.96, <0.0001
[O3] x Line
1.87, 0.076
1.77, 0.094
3.12, 0.004
5.51, <0.0001
5.76, <0.0001
1.23, 0.296
2.29, 0.028
2.58, 0.014
Hybrid Lines
A

gs



A (mol m-2 s-1)
gs (mol m-2s-1)
[O3]
0.00, 0.965
0.00, 0.953
55.9, 0.017
66.4, 0.015
61.8, 0.016
1.82, 0.310
4.15, 0.179
4.72, 0.095
Line
1.04, 0.429
0.39, 0.902
1.52, 0.202
1.67, 0.157
4.50, 0.002
0.77, 0.618
1.80, 0.128
3.98, 0.004
[O3] x Line
0.58, 0.765
1.19, 0.340
5.72, 0.0004
8.16, <0.0001
10.4, <0.0001
1.60, 0.177
1.97, 0.095
2.36, 0.050
yo
yo
28
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Table 4. The percentage change in elevated [O3] for the sum of midday photosynthesis (A) and
average stomatal conductance (gs) of the leaf subtending the ear measured during grain filling in
2015. Data and quadratic fits for A and gs in inbred and hybrid lines are shown in Figs. 5-8.
Line
A
gs
B73
-21%
0%
C123
-59%
-13%
CML333
0%
0%
Hp301
-17%
0%
IL14H
-34%
0%
M37W
0%
0%
Mo17
-16%
0%
MS71
-46%
-5%
NC338
-2%
-12%
OH43
-28%
+14%
B73xC123
-39%
-31%
B73xCML333
-22%
0%
B73xHP301
-32%
0%
B73xIL14H
-39%
0%
B73xM37W
-13%
0%
B73xMo17
-43%
0%
B73xMS71
-44%
-43%
B73xOH43
-28%
-27%
29
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
FIGURE LEGENDS
Figure 1. The diurnal response of net assimilation (A) and stomatal conductance (gs) of maize
inbred line B73 in 2013. Measurements were made on the leaf subtending the ear on three days
of the year (DOY). The number of days from anthesis is shown in parenthesis after measurement
DOY. The conditions in the leaf cuvette including leaf temperature (Tleaf), vapor pressure deficit
(VPD), and photosynthetically active radiation (PAR) are given for each date in panels a, b and
c. Diurnal A is shown for each DOY in panels d, e and f, and gs is shown in panels g, h and i. The
least squared means ± 1 standard error are shown. The p-value represents the O3 effect for each
DOY.
Figure 2. The diurnal response of net assimilation (A) and stomatal conductance (gs) of maize
inbred line B73 in 2014. Measurements were made on the leaf subtending the ear on four days of
the year (DOY). The number of days from anthesis is shown in parenthesis after measurement
DOY. The conditions in the leaf cuvette including leaf temperature (Tleaf), vapor pressure deficit
(VPD), and photosynthetically active radiation (PAR) are given for each date in panels a, b, c
and d. Diurnal A is shown for each DOY in panels e, f, g and h, and gs is shown in panels i, j, k
and l. The least squared means ± 1 standard error are shown. The p-value represents the O3 effect
for each DOY.
Figure 3. Maximum carboxylation capacity of PEPC (Vpmax) (a, b) and CO2-saturated
photosynthetic rate (Vmax) (c, d) of maize inbred line B73 measured in 2013 (a, c) and 2014 (b,
d). Measurements were made on the leaf subtending the ear. The least squared means ± 1
30
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
standard error are shown. Significant differences between ambient and elevated [O3] (p<0.05) for
individual days are indicated with an asterisk.
Figure 4. Summary of A/ci response curves (solid lines) and CO2 supply functions (dashed lines)
for maize inbred line B73 grown at ambient [O3] (black lines) and elevated [O3] (grey lines). The
supply functions show the maximum and minimum ci observed at midday in the field during
diurnal measurements of A, and the points on the curves show the mean observed ci.
Measurements were made on the leaf subtending the ear shortly after anthesis (a, b) and ~4-5
weeks after anthesis (c, d). Estimates of mean stomatal limitation (l) are reported in each panel
for ambient and elevated [O3].
Figure 5. Photosynthetic rate (A) of the leaf subtending the ear measured during grain filling in
2015 for 10 inbred lines grown at ambient (open circles) and elevated [O3] (filled circles). The
best fit quadratic curves for ambient (dashed line) and elevated [O3] (solid line) are shown if the
statistical test revealed that there was a better fit to the data using two quadratic curves (i.e., one
for each treatment) rather than a single curve for all of the data. Grey lines represent the best
quadratic fit through all of the data when the statistical test revealed no difference between
ambient and elevated [O3]. Each point represents the mean value for an individual replicate ring
for each measurement date. Parameters describing these curves (yo, , ) are reported in
Supplemental Table 1.
Figure 6. Stomatal conductance (gs) of the leaf subtending the ear measured during grain filling
in 2015 for 10 inbred lines grown at ambient (open circles) and elevated [O3] (filled circles). The
31
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
best fit quadratic curves for ambient (dashed line) and elevated [O3] (solid line) are shown if the
statistical test revealed that there was a better fit to the data using two quadratic curves (i.e., one
for each treatment) rather than a single curve for all of the data. Grey lines represent the best
quadratic fit through all of the data when the statistical test revealed no difference between
ambient and elevated [O3]. Each point represents the mean value for an individual replicate ring
for each measurement date. Parameters describing these curves (yo, , ) are reported in
Supplemental Table 1.
Figure 7. Photosynthetic rate (A) of the leaf subtending the ear measured during grain filling in
2015 for 8 hybrid lines grown at ambient (open circles) and elevated [O3] (filled circles). The
best fit quadratic curves for ambient (dashed line) and elevated [O3] (solid line) are shown if the
statistical test revealed that there was a better fit to the data using two quadratic curves (i.e., one
for each treatment) rather than a single curve for all of the data. Grey lines represent the best
quadratic fit through all of the data when the statistical test revealed no difference between
ambient and elevated [O3]. Each point represents the mean value for an individual replicate ring
for each measurement date. Parameters describing these curves (yo, , ) are reported in
Supplemental Table 1.
Figure 8. Stomatal conductance (gs) of the leaf subtending the ear measured during grain filling
in 2015 for 8 hybrid lines grown at ambient (open circles) and elevated [O3] (filled circles). The
best fit quadratic curves for ambient (dashed line) and elevated [O3] (solid line) are shown if the
statistical test revealed that there was a better fit to the data using two quadratic curves (i.e., one
for each treatment) rather than a single curve for all of the data. Grey lines represent the best
32
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
quadratic fit through all of the data when the statistical test revealed no difference between
ambient and elevated [O3]. Each point represents the mean value for an individual replicate ring
for each measurement date. Parameters describing these curves (yo, , ) are reported in
Supplemental Table 1.
Supplemental Figure 1. Seasonal maximum and minimum temperatures and precipitation in
2013, 2014 and 2015 measured at the FACE experimental site in Champaign, IL.
Supplemental Figure 2. Instantaneous water use efficiency (iWUE = A/gs) of the leaf
subtending the ear measured during grain filling in 2015 for 10 inbred lines grown at ambient
(open circles) and elevated [O3] (filled circles).
Supplemental Figure 3. Instantaneous water use efficiency (iWUE = A/gs) of the leaf
subtending the ear measured during grain filling in 2015 for 8 hybrid lines grown at ambient
(open circles) and elevated [O3] (filled circles).
33
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 1.
34
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2.
35
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 3.
36
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 4.
37
Figure 5.
38
Figure 6.
39
Figure 7.
40
Figure 8.
41
bioRxiv preprint first posted online Jun. 23, 2017; doi: http://dx.doi.org/10.1101/154104. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplemental Table 1. Parameters describing the quadratic function describing the decline in A
and gs over time in the leaf subtending the ear, for 10 inbred lines and 8 hybrid lines grown at
ambient and elevated [O3]. Significant differences in the quadratic fits between ambient and
elevated [O3] for each inbred or hybrid line are shown in bold font.
Line
B73
C123
CML333
HP301
IL14H
M37W
Mo17
MS71
NC338
OH43
B73 x C123
B73 x CML333
B73 x HP301
B73 x IL14H
B73 x M37W
B73 x Mo17
B73 x MS71
B73 x OH43
[O3]
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
yo
25.6
35.9
10.2
49.4
30.8
30.8
24.5
29.6
61.5
63.4
22.3
22.3
25.9
16.6
48.6
60.4
24.8
30.6
20.0
28.8
4.72
17.0
15.7
16.7
6.35
15.5
7.52
24.0
15.4
16.8
3.37
24.1
1.02
21.4
-4.85
24.4
A

0.91
-0.15
1.99
-1.99
0.22
0.22
0.80
0.13
-1.89
-2.62
0.0077
0.0077
0.30
0.75
-1.19
-2.47
0.28
0.012
0.97
0.28
1.28
0.10
0.41
0.34
0.93
0.19
0.98
-0.22
0.19
0.28
1.27
-0.41
1.42
-0.28
1.71
-0.19

-0.027
-0.011
-0.045
0.020
-0.014
-0.014
-0.026
-0.015
0.011
0.026
-0.0046
-0.0046
-0.015
-0.022
0.0035
0.025
-0.011
-0.010
-0.022
-0.014
-0.026
-0.0089
-0.0078
-0.0097
-0.017
-0.0082
-0.020
-0.0054
-0.0038
-0.0083
-0.025
-0.0014
-0.028
-0.0031
-0.030
-0.0053
yo
0.16
0.16
0.026
0.37
0.19
0.19
0.14
0.14
0.59
0.59
0.13
0.13
0.073
0.073
0.37
0.42
0.096
0.047
0.17
0.17
0.18
0.099
0.11
0.11
0.0063
0.0063
0.041
0.041
0.13
0.13
0.075
0.075
0.033
0.071
-0.15
0.17
gs

0.0056
0.0056
0.015
-0.014
0.00078
0.00078
0.0052
0.0052
-0.025
-0.025
0.00087
0.00087
0.0064
0.0064
-0.011
-0.018
0.0046
0.0084
0.0025
0.0025
0.0071
0.0066
0.0051
0.0051
0.014
0.014
0.012
0.012
0.0022
0.0022
0.010
0.010
0.0116
0.0037
0.023
0.00060

-0.00018
-0.00018
-0.00037
0.00013
-0.00007
-0.00007
-0.00017
-0.00017
0.00025
0.00025
-0.00004
-0.00004
-0.00016
-0.00016
0.000066
0.00020
-0.00012
-0.00019
-0.00009
-0.00009
-0.00021
-0.00017
-0.00010
-0.00010
-0.00024
-0.00024
-0.00025
-0.00025
-0.00005
-0.00005
-0.00023
-0.00023
-0.00024
-0.00010
-0.00039
-0.00007
42
Supplemental Figure 2.
43
Supplemental Figure 3.
44